MicroRNA Profiling Revealed the Mechanism of Enhanced Cold Resistance by Grafting in Melon (Cucumis melo L.)

Grafting is widely used to improve the resistance to abiotic stresses in cucurbit plants, but the effect and molecular mechanism of grafting on cold stress are still unknown in melon. In this study, phenotypic characteristics, physiological indexes, small-RNA sequencing and expression analyses were performed on grafted plants with pumpkin rootstock (PG) and self-grafted plants (SG) to explore the mechanism of changed cold tolerance by grafting in melon. Compared with SG plants, the cold tolerance was obviously enhanced, the malondialdehyde (MDA) content was significantly decreased and the activities of antioxidant enzymes (superoxide dismutase, SOD; catalase, CAT; peroxidase, POD) were significantly increased in PG plants. Depend on differentially expressed miRNA (DEM) identification and expression pattern analyses, cme-miR156b, cme-miR156f and chr07_30026 were thought to play a key role in enhancing low-temperature resistance resulting from grafting. Subsequently, 24, 37 and 17 target genes of cme-miR156b, cme-miR156f and chr07_30026 were respectively predicted, and 21 target genes were co-regulated by cme-miR156b and cme-miR156f. Among these 57 unique target genes, the putative promoter of 13 target genes contained the low-temperature responsive (LTR) cis-acting element. The results of qRT-PCR indicated that six target genes (MELO3C002370, MELO3C009217, MELO3C018972, MELO3C016713, MELO3C012858 and MELO3C000732) displayed the opposite expression pattern to their corresponding miRNAs. Furthermore, MELO3C002370, MELO3C016713 and MELO3C012858 were significantly downregulated in cold-resistant cultivars and upregulated in cold-sensitive varieties after cold stimulus, and they acted as the key negative regulators of low-temperature response in melon. This study revealed three key miRNAs and three putative target genes involved in the cold tolerance of melon and provided a molecular basis underlying how grafting improved the low-temperature resistance of melon plants.


Introduction
The melon (Cucumis melo L.) is an annual plant of the Cucurbitaceae family, and it is an important economic crop.Melons prefer warm climates and have a certain degree of resistance to heat but are very sensitive to cold [1].Temperature is an important environmental factor affecting melon growth and the quality of fruit produced.The optimal growth temperature of melon is 25-35 • C/15-18 • C for the day/night periods [2,3].The optimum temperature for melon also varies slightly between different varieties and changes slightly during the growth and development of the plants.However, melons will be subjected to severe cold stress below 15 • C, and the damage caused is irreversible when the temperature is below 6 • C for 5-6 days [4].Therefore, low temperature is the main abiotic stress that limits the production and quality of melons in cool regions, such as northern and northeastern China, especially during the winter and the spring.
When the plants were subjected to abiotic stresses, abundant reactive oxygen species (ROS) were produced, which led to lipid peroxidation of cell membranes and promoted the generation of malondialdehyde (MDA).Excessive accumulation of MDA resulted in further damage to the cell membranes, and the content of MDA was an important indicator reflecting the damage degree of the cell membrane and plant resistance to abiotic stresses [5,6].Low-temperature stress is known to induce high production of ROS and MDA, which damage the cell membrane and organelles [7,8].In addition, low temperature also limits plant photosynthesis and respiration and leads to reduced growth or even death of melon plants [4,9].
Grafting acts as an important technique and is widely applied in horticultural crops.Grafting can maintain the excellent characteristics of the scion and effectively enhance resistance to multiple biotic and abiotic stresses in grafted plants relying on the adaptable roots of rootstocks [10][11][12][13].Therefore, grafting is routinely practiced to improve the cold resistance of numerous vegetables, especially cucurbits [14][15][16].After the application of grafting, the root vitality is increased, the cell membrane stability is enhanced, multiple antioxidant enzyme activities are induced and the impact of low temperature on photosynthesis is reduced, so the cold resistance is significantly improved in grafted plants [17].For example, the relative electrolyte leakage and malondialdehyde content are significantly decreased in cucumber seedlings grafted onto luffa rootstocks compared with self-grafted plants [16].In addition, the photochemical efficiency of cucumber plants grafted onto the gourd rootstock is significantly increased, which further leads to increased low-temperature tolerance in grafted seedlings [18].In watermelon, the cold resistance of grafted plants is significantly enhanced by the application of pumpkin rootstock, and the lipid peroxidation and protein oxidation processes are restricted in grafted plants [16,19].In melon, grafting also acts as an effective manner of improving the abiotic stresses such as heavy metal stress and salt stresses [20,21].Grafting can change the contents of sugars and organic acid and regulate multiple enzyme activities such as sucrose phosphate synthase and POD in melon [20,22].
MicroRNA (miRNA) is a type of endogenous, non-coding, single-stranded small RNA with the length of 19 to 24 nt [23].MiRNAs have been reported to widely exist in plants and participate in various growth and development processes such as seed germination, flower organ development, fruit enlargement and ripening [24][25][26][27][28]. Expectedly, miRNAs also play an important role in resisting abiotic stresses including drought, salt, high temperature or cold [29][30][31][32].In Arabidopsis, miRNAs are first confirmed to respond to low temperature, and the cold tolerance is significantly improved in Arabidopsis plants upon the overexpression of miR397, miR402 or miR408 [33][34][35].In rice, the constitutive expression of miR319b also results in enhanced low-temperature resistance [36].Similarly, the overexpression of sha-miR319d increases the cold tolerance of tomato [37].However, the cold tolerance of tomato plants is significantly decreased with the upregulation of sly-miR156e-3p or miR164a [38,39].In apple, the survival rate of plants overexpressing Mdm-miR160e is less than that of wild-type plants under low-temperature stress of 0 • C for 8 h [40].In turnip, Bra-novel-miR3936-5p is induced but miR319e and miR166m-2 are suppressed by cold stress of −4 • C for 8 h [41].These results suggest that the responses of miRNAs to low temperature are not conserved in different species, and miRNAs include positive and negative regulators of cold stress.
MiRNAs carry out their function by regulating the expression of target genes [42].There is a negative regulatory relationship between miRNAs and their target genes, and miRNAs are able to cleave and degrade their target genes by a complete complementary combination manner [43,44].For example, the overexpression of OsmiR156 enhances the cold tolerance of rice by suppressing the transcripts of its target gene OsSPL3 (squamosa promoter-binding protein-like 3) [25,45].Mdm-miR160 plays a negative role in freezing tolerance, but MdARF17 is targeted by Mdm-miR160 and acts as a positive regulator of freezing tolerance in apple [40].Similarly, an miR169 and NF-YA/HAP2 module is proposed to participate in the low-temperature response of purple alfalfa by degradome sequencing analysis [46].
CBFs (C-repeat binding factors) are important regulators for the cold stress response of plants and belong to the DREB subfamily of AP2/ERF (APETALA2/ETHYLENE-responsive factor) family [47].In Arabidopsis, the transcripts of CBF genes are rapidly induced after 15-30 min of cold treatment and peak at 1-3 h of cold stress [48].The overexpression of CBF3 increases the contents of proline and total soluble sugars and further improves the cold resistance of Arabidopsis [49].The upregulation of BnCBF5 and BnCBF17 leads to depressed leaf electrolyte leakage and enhanced chilling tolerance compared with wild-type Brassica napus [50].CBFs regulate cold tolerance through binding to C-repeat/dehydration responsive element (CRT/DRE, A/GCCGAC) cis-acting motifs in the promoters of coldregulated genes (CORs) [51].In sweet potato, IbCBF3 induces the transcripts of IbCOR27, IbCOR314 and IbCOR413 by binding to CRT/DRE elements in the promoters of COR genes.The overexpression of IbCBF3 enhances the cold stress tolerance of sweet potato and results in decreased contents of MDA and H 2 O 2 under low-temperature stress [52].In addition, cold response is also regulated by multiple transcription factors such as WRKY, MYB, NAC and the above-mentioned SPLs [53][54][55][56].Ectopic expression of cucumber CsWRKY46 into Arabidopsis leads to higher proline accumulation, less electrolyte leakage and lower MDA levels than that in the wild type, and the survival rate of seedlings is significantly improved in CsWRKY46-overexpressing plants after freezing treatment [57].In apple, MYB308L positively regulates anthocyanin accumulation and enhances the low-temperature tolerance [58].In pepper, CaNAC2 is significantly induced by low temperature, and the inhibition of CaNAC2 increases the sensitivity of pepper seedlings to low temperatures [59].
In melon production, grafting can effectively improve the resistance of plants to various abiotic stresses [20,21], but the effect and molecular mechanism of grafting on low-temperature stress are still not fully clear.MiRNAs and corresponding target genes actively participate in low-temperature stress responses [25,42].However, the key miRNAs and target genes responding to low-temperature stress in melon are still poorly understood, and the regulatory networks between miRNAs and their target genes need further study.Therefore, phenotypic characteristics, physiological indexes, small-RNA sequencing and expression analyses were performed in SG and PG plants in this study to explore the relationship between grafting and low-temperature response and to investigate the mechanism underlying how the plants resist low temperatures in melon.

Grafting with Pumpkin Rootstock Enhanced the Plant Resistance to Low Temperatures in Melon
In melon production, grafting is an effective technique to enhance the plant resistance to multiple diseases and abiotic stresses [60].In order to understand the effect of grafting on the low-temperature response of melon plants, morphological characteristics of selfgrafted seedlings (SG) and seedlings grafted with pumpkin rootstock 'YinGuang' (PG) were investigated under low-temperature stress at 4 • C (Figure 1a).Both SG and PG plants dehydrated and wilted with the extension of low temperature, but the PG plants showed greater resistance to low temperature than SG plants (Figure 1a).After 12 h of low temperature, the phenotype differences were apparent, and self-grafted seedlings showed more severe wilting compared with PG plants (Figure 1a).Therefore, grafting with pumpkin rootstock effectively improved the resistance of melon plants to low-temperature stress.
Subsequently, the content of MDA and the activities of antioxidases (SOD, CAT and POD) were detected to understand the phenotype difference between SG and PG plants under low-temperature stress (Figure 1b-e).The MDA content in SG plants was distinctly induced by low temperature from 0 to 12 h but decreased at 16 h after cold treatment (Figure 1b).In PG plants, the MDA content was also significantly increased from 0 to 8 h but visibly declined at 12 h of low-temperature treatment (Figure 1b).In summary, the content of MDA in PG seedlings was always lower and decreased earlier than that in SG seedlings under low-temperature stress (Figure 1b).The activities of three antioxidant enzymes (SOD, CAT and POD) were significantly higher in PG seedlings than in SG seedlings at all detected time points after low-temperature treatment (Figure 1c-e).The variation tendency of SOD and CAT activities in SG seedlings was similar to that in PG seedlings (Figure 1c,d), but there was a difference in the initial POD content between SG and PG seedlings, which may result from the effect of grafting with pumpkin rootstock (Figure 1e).These results suggested that grafting with pumpkin rootstock could effectively decrease the MDA content and increase the activities of SOD, CAT and POD in melon seedlings under low-temperature stress.

Small-RNA Signatures of SG and PG Seedlings under Low-Temperature Stress
MiRNAs have been reported to widely exist in various plants and play an important role in responding to low-temperature stress [24].To investigate the effect of miRNAs on the resistance of grafted melon plants to low-temperature stress, small-RNA sequencing was performed on SG and PG seedlings treated with 4 • C for 12 h (L-SG/L-PG), and normal temperature (25 • C) was used for control (N-SG/N-PG).Three biological replicates were performed for each treatment, and thus 12 libraries were produced (Table S1).For each sample, 10.29 to 18.55 million raw reads were generated by high-throughput sequencing.After quality control of the original sequencing data, 9.97 to 18.14 million clean reads were obtained.According to the sRNA length characteristics, 18 nt to 32 nt (nucleotide) reads were determined as useful reads, and 9.33 to 17.27 million useful reads were obtained (Table S1).The length for most of small RNAs ranged from 20 nt to 25 nt, and the small RNAs with the length of 24 nt accounted for the largest proportion in all 12 libraries (Figure 2a).Finally, 7.00 to 13.01 million useful reads were mapped to the melon genome (http://cucurbitgenomics.org/organism/18 (accessed on 6 January 2023)) by Bowtie software (v1.2.3) (Table S1) [61].The known miRNAs were acquired by miRBase and Rfam databases, and the novel miRNAs were identified by miRDeep2 software (v2.0.1.3)[62,63].Meanwhile, repeated sequences and ncRNAs such as ribosomal RNA (rRNA), transport RNA (tRNA), small intracellular RNA (snRNA) and small nucleolar RNA (snoRNA) were filtered.In total, 105 to 112 known miRNAs and 104 to 106 novel miRNAs were identified for each sample (Table 1).After removing the repeated miRNAs from each library, 222 unique miRNAs were finally acquired and included 116 known miRNAs and 106 novel miRNAs (Table S2).The 116 known miRNAs belonged to 28 miRNA families in which the number of miRNAs in the miR156 (ten) and miR169 (twenty) families were the largest (Table S2).The square of Pearson's correlation coefficient (R 2 ) between biological replicates in the same treatment was obviously higher than that in different treatments (Figure 2b).Principal component analysis (PCA) showed that samples from the same treatment were distinctly clustered together and clearly distinguished from samples of other treatments (Figure 2c).These results ensured the reliability of subsequent differentially expressed miRNA (DEM) identification, and suggested that the miRNAs profiles were different in N-SG, N-PG, L-SG and L-PG.
In addition, the miRNA expression profiles of N-PG and L-PG were compared, and seven miRNAs were differentially expressed.Compared with N-PG plants, five miRNAs (cme-miR156b, cme-miR156f, chr00_3087, chr07_30026 and chr11_40459) were significantly induced, whereas cme-miR167c and chr00_2803 were significantly downregulated in L-PG plants (Table S3).Finally, the common DEMs (cme-miR156b, cme-miR156f, chr00_3087 and chr07_30026) between the comparison set of N-PG vs. L-PG and the above-mentioned 12 DEMs were thought to play a key role in enhancing the low-temperature resistance of grafted plants with pumpkin rootstock.
In order to validate the DEM results, independent samples were collected in the same manner as in the DEM analysis, and eight DEMs were randomly chosen to perform the qRT-PCR analysis (Figure 3).The expression patterns of the selected DEMs detected by qRT-PCR were consistent with those from sRNA-seq data (Figure 3).The Pearson's correlation coefficient between sRNA-seq and qRT-PCR data was 0.82, indicating highly reliable sRNA-seq results.

Expression Patterns of Four Key DEMs under Low-Temperature Stress and Target Gene Prediction
In order to explore the response of four key DEMs to low-temperature stress, the expression levels of cme-miR156b, cme-miR156f, chr00_3087 and chr07_30026 were investigated in SG and PG seedlings under different low-temperature durations (Figure 4).In SG seedlings, the expression levels of cme-miR156b, cme-miR156f and chr00_3087 were significantly increased at 4 h of cold treatment and reached the peak value at 8 h of lowtemperature treatment but rapidly decreased after 12 h and then 16 h of low-temperature stress (Figure 4a,b,d).However, chr07_30026 was significantly suppressed by cold treatment at 4 h and then exhibited increased expression compared with 0HAC (0 h after cold treatment) (Figure 4c).In PG seedlings, cme-miR156b and cme-miR156f were also significantly increased after low-temperature stress (4, 8 and 12 h) and returned to a similar level as 0HAC (Figure 4e,f).Chr07_30026 was also significantly induced at 4 h of cold treatment and then declined with subsequent low-temperature treatment in PG seedlings (Figure 4g).However, there was no significant difference in the expression level of chr00_3087 from 0 to 12 h of low-temperature stress, though slight upregulation was observed (Figure 4h).These results suggested that cme-miR156b, cme-miR156f and chr07_30026 displayed an active response to low-temperature stress.The functions of miRNAs were mainly dependent on post-transcriptional regulation of their target genes [64].An miRNA was able to regulate multiple target genes, and one gene may also be regulated by different miRNAs [65].In this study, 7556 and 5649 target genes were respectively predicted for 116 known miRNAs and 106 novel miRNAs by the online software TargetFinder (v5.8) (https://targetfinder.org/(accessed on 13 November 2022)) (Table S4) [66].In order to explore the regulators involved in low-temperature resistance mediated by grafting, the target genes of cme-miR156b, cme-miR156f and chr07_30026 were identified.In total, 24 and 37 target genes were predicted for cme-miR156b and cme-miR156f, of which 21 target genes were co-regulated by cme-miR156b and cme-miR156f (Table S5).The number of target genes was 17 for the novel miRNA chr07_30026 (Table S5).In total, 57 unique target genes regulated by cme-miR156b, cme-miR156f and chr07_30026 were predicted (Table S5).

Expression Pattern Analyses of Key Target Genes under Low-Temperature Stress
The presence of LTR cis-acting elements plays an important role in regulating gene expression when plants encounter cold stimuli [67].According to promoter sequence analysis, 13 out of 57 target genes possessed LTR cis-acting elements (Figure 5a), and the expression patterns of these 13 target genes were analyzed by qRT-PCR under low-temperature stress.In SG plants, the expression levels of cme-miR156b and cme-miR156f were slightly reduced, and the expression level of chr07_30026 was slightly induced by low temperature in SG plants (Figure 5b-d).However, cme-miR156b, cme-miR156f and chr07_30026 were significantly induced by cold in PG plants (Figure 5b-d).The putative target genes MELO3C002370, MELO3C009217 and MELO3C018972 exhibited the opposite expression patterns to cme-miR156b and cme-miR156f and were increased and decreased in L-SG and L-PG, respectively (Figure 5e,i,j).Similarly, MELO3C016713, MELO3C012858 and MELO3C000732 showed the inverse expression pattern to chr07_30026 and were suppressed by low-temperature stress in SG and PG plants (Figure 5m-o).There were no opposite expres-sion patterns between the other seven putative target genes (MELO3C023559, MELO3C022318, MELO3C002159, MELO3C026599, MELO3C015061, MELO3C012289 and MELO3C014018) and relevant miRNAs (cme-miR156b, cme-miR156f and chr07_30026) (Figure 5).Therefore, MELO3C002370, MELO3C009217, MELO3C018972, MELO3C016713, MELO3C012858 and MELO3C000732 were considered the key candidate genes in regulating grafting-enhanced low-temperature resistance.(a) LTR (yellow triangles) cis-acting element analysis of target genes.(b-q) Expression analyses of three miRNAs (b-d) and their target genes (e-q) in SG (blue columns) and PG (yellow columns) plants after 12 h of normal or cold treatment.Black triangles, circles and rectangles represent the genes targeted by cme-miR156b, cme-miR156f and chr07_30026, respectively.Lowercase letters above the columns represent the significant differences at p < 0.05 (Duncan's test).
Subsequently, the expression levels of MELO3C002370, MELO3C009217, MELO3C018972, MELO3C016713, MELO3C012858 and MELO3C000732 were investigated in the dynamic low-temperature process of SG and PG plants (Figure 6a-l).In SG plants, MELO3C002370, MELO3C009217 and MELO3C018972 were significantly downregulated after 4 h of low temperature, but an unexpected upregulation was observed at 12 h of low temperature (Figure 6a-c).However, the downregulation of MELO3C016713, MELO3C012858 and MELO3C000732 was relatively delayed compared with that of MELO3C002370, MELO3C009217 and MELO3C018972 and significantly decreased after 8 h of low temperature (Figure 6d-f).In PG plants, all six above-mentioned candidate genes except for MELO3C018972 were significantly suppressed from 4 h of low temperature (Figure 6g-l).The downregulation of MELO3C016713, MELO3C012858 and MELO3C000732 in PG plants occurred earlier than that in SG plants, and their expression levels were similar to the 0HAC until 16 h of low temperature (Figure 6j-l).The transcripts of MELO3C009217 were significantly increased at 12 h and close to 0HAC at 16 h of low temperature (Figure 6h).It is worth noting that the expression level of MELO3C002370 remained at a low level after 16 h of low-temperature treatment in PG plants (Figure 6g).MELO3C018972 was significantly downregulated at 8 h of low temperature and showed a later downregulation in PG plants compared with that in SG plants (Figure 6c,i).Therefore, MELO3C009217, MELO3C016713, MELO3C012858, MELO3C000732 and especially MELO3C002370 may act as the major negative regulators in enhancing the cold resistance of PG plants.
In addition, the expression levels of MELO3C002370, MELO3C009217, MELO3C018972, MELO3C016713, MELO3C012858 and MELO3C000732 were investigated in several cultivars of melon with different low-temperature resistance (strong, TZ4 and QM1; middle, QM2; weak, YZ9 and TM) (Figure 7a).Compared with control, the expression levels of MELO3C002370, MELO3C016713 and MELO3C012858 were significantly decreased in TZ4 and QM1 and significantly increased in YZ9 and TM under low-temperature stress (Figure 7b-d).In QM2, there was no significant difference in the transcripts of MELO3C002370 and MELO3C016713, but MELO3C012858 was significantly reduced after low-temperature stress (Figure 7b-d).The transcripts of MELO3C009217, MELO3C018972 and MELO3C000732 also changed in several melons, but their expression levels were not perfectly consistent with the low-temperature resistance of given cultivars (Figure 7e-g).These results further suggested that MELO3C002370, MELO3C016713 and MELO3C012858 were the key negative regulators of low-temperature response in melon.MELO3C002370, MELO3C016713 and MELO3C012858, respectively, were the homologs of SPL13, ABA receptor GCR2 and RIC7 by BLAST searches in the Arabidopsis Information Resource (http://www.arabidopsis.org/(accessed on 31 March 2005)) using their full-length amino acid sequences (Table 2).

Discussion
The stress resistance of plants is closely linked to the stability of the cell membrane and the accumulation of osmotic substances.When plants experience abiotic stresses, the permeability of the cell membrane increases, leading to the production of a large quantity of toxic substances such as MDA and ROS [7,8].Fortunately, grafting can effectively reduce the accumulation of toxic substances, promote the production of osmotic substances such as proline and soluble protein, and enhance the antioxidant enzyme activity to improve the stress resistance of grafted plants [60,68].At present, the effect and mechanism of grafting on cold tolerance are still not clear in melon.This study explored the differences in morphology and physiological indexes between PG and SG plants under low-temperature stress.Compared with SG plants, grafted seedlings with pumpkin rootstock showed enhanced cold tolerance, less MDA content and higher activities of SOD, CAT and POD (Figure 1).These results indicated that grafting with appropriate rootstock could effectively improve the resistance of melon plants to cold stress and significantly alter the levels of physiological indexes related to low-temperature response.
In order to investigate the mechanism of enhanced cold tolerance resulting from grafting, small-RNA sequencing was performed on SG and PG plants under low-temperature (L-SG/L-PG) and normal-temperature (N-SG/N-PG) conditions.Combined with DEM identification and expression pattern analyses, cme-miR156b, cme-miR156f and chr07_30026 were identified as the major regulators of low-temperature response mediated by grafting.Previous studies have indicated that miR156 is extensively involved in the response to cold stress [25,69].In sugarcane, miR156 is significantly induced by low-temperature stress, and the ectopic expression of sugarcane miR156 leads to better growth status, lower ROS level, and higher anthocyanin content in tobacco leaves under cold conditions [70].In rice, OsmiR156 also positively regulates the cold stress tolerance by increasing cell viability and growth rate after low-temperature treatment [45].However, sly-miR156e-3p of tomato acts as a negative regulator of cold tolerance, and the overexpression of sly-miR156e-3p displays more severe leaf wilting, lower maximal photochemical efficiency of PSII and higher relative electrolyte leakage compared with the wild type [38].Therefore, the function of miR156 is not conserved in different species.In this study, cme-miR156b and cme-miR156f were significantly induced by low-temperature stress in PG and SG plants (Figure 4a,b,e,f), which suggested that cme-miR156b and cme-miR156f acted as the positive regulators of cold stress in melon.At present, the functions of cme-miR156b and cme-miR156f in melon are still unknown, and their importance in responding to low-temperature stress remain to be clarified.In addition, a novel miRNA, chr07_30026, was also significantly increased after cold treatment in PG plants, though chr07_30026 was suppressed by low temperature at 4 h in SG plants (Figure 4c,g).
MiRNAs mainly work by regulating the expression of target genes [71].In tomato, sha-miR319d improves the cold tolerance of plants by inhibiting the transcription of GAMYBlike 1 [37].Similarly, OsmiR528 inhibits the expression of OsMYB30, which negatively regulates BMY4 (β-amylase 4), BMY6 and BMY10 to increase the activity of β-amylase and further improves the low-temperature tolerance of rice [72].In this study, 24, 37 and 17 target genes of cme-miR156b, cme-miR156f and chr07_30026 were respectively pre-dicted, and 13 target genes with the LTR cis-acting element were chosen to further analyze (Figure 5a).In total, six target genes (MELO3C002370, MELO3C009217, MELO3C018972, MELO3C016713, MELO3C012858 and MELO3C000732) showed the opposite expression pattern to their corresponding miRNAs (Figure 5b-q).In addition, the expression patterns of the above-mentioned six target genes under low-temperature stress were investigated in several cultivars of melon with different low-temperature resistance, and three target genes (MELO3C002370, MELO3C016713 and MELO3C012858) were downregulated in cold-resistant cultivars and upregulated in cold-sensitive varieties (Figure 7).These results suggested that MELO3C002370, MELO3C016713 and MELO3C012858 acted as the key negative regulators of cold stress response mediated by grafting in melon.
The results of homologous alignment using the full-length amino acid sequence showed that MELO3C002370, MELO3C016713 and MELO3C012858 were the homologs of Arabidopsis SPL13, ABA receptor GCR2 and RIC7, respectively (Table 2).However, the functions of putative CmSPL13 (MELO3C002370), CmGCR2 (MELO3C016713) and CmRIC7 (MELO3C012858) are still not clear in melon.In Arabidopsis, SPL13 promotes floral transition and regulates adult leaf morphology by directly repressing BLADE-ON-PETIOLE 1 (BOP1) and BOP2 [73].In addition, the homologs of SPL13 are also involved in counteracting drought, heat and flooding stresses in Medicago sativa [74][75][76].At present, the function of SPL13 in cold stress response is still unknown.GCR2 functions as a G-protein-coupled receptor (GPCR) for ABA, but no obvious morphological defect is observed in loss-offunction alleles of Arabidopsis GCR2, which may result from the subtle phenotypes of GCR2, the functional redundancy with other genes or the exhibition of phenotypes only under certain conditions [77,78].RIC7 has been found to negatively regulate ABA-induced stomatal closure.Compared with the wild type, plants with overexpression of RIC7 exhibit larger stomatal apertures, accumulate less H 2 O 2 and reduce the expression levels of genes related to ROS generation after ABA treatment [79].
Melons prefer warm climates and have a certain degree of resistance to heat but are very sensitive to cold.It is urgent to breed a melon variety with strong cold tolerance, but the molecular mechanism for the low-temperature response of melon is still unclear.According to the existing research and the results of this study, cme-miR156b, cme-miR156f and their common target gene CmSPL13 were considered the key regulators for lowtemperature response during grafting in melon.In the future, the function and mechanism of the cme-miR156b/cme-miR156f -CmSPL13 module in regulating cold tolerance will be explored to further understand the interaction between melon plants and the environment.The results of this study provided a theoretical basis for breeding new melon varieties with high cold tolerance, and it is of great significance for the high-quality and efficient production of melon.

Plant Materials and Treatments
The melon variety 'Boyang61' was used as the scion, and 'Yin Guang' (Cucurbita maxima Duch.) was used as the rootstock.The controls were self-grafted plants of 'Boyang61' (one plant of 'Boyang61' was used as the scion and grafted onto another 'Boyang61' rootstock).When the seedlings of rootstocks reached the one-true-leaf stage and the cotyledons of scions displayed, grafting was performed using the cut-grafting method [80].The grafted seedlings were grown in the greenhouse of Hebei Agricultural University under 25-30 • C and 85-100% relative humidity for approximately 7 days [81].After the complete healing of grafted seedlings, the plants were transferred to standard management practices.At the 4-true-leaves stage (about 35 days after grafting), the apex of grafted plants was collected at 0 h, 4 h, 8 h, 12 h and 16 h after cold treatment (4 • C).The samples were rapidly frozen by liquid nitrogen and stored at −80 • C for physiological indicator determination and small-RNA-seq library construction.

Determination of Physiological Indicators
Malondialdehyde (MDA) was estimated from the third true leaves of PG and SG plants using 5% trichloroacetic acid (TCA) according to previous methods [8].A 2 mL aliquot of enzyme extract was mixed with 10% TCA containing 0.65% (w/v) thiobarbituric acid (TBA), placed in a water bath at 100 • C for 30 min and then immediately cooled to stop the reaction.The reaction mixture was centrifuged for 10 min at 800× g, and the optical density (OD) value was measured at 532 nm, 600 nm and 450 nm.

Small-RNA Library Construction and Small-RNA Sequencing
Total RNA was isolated from the leaves of PG and SG plants after low-temperature treatment for 0 h (control) and 12 h and checked on 1% agarose gels to avoid possible degradation and contamination.A NanoDrop 2000 photometer spectrophotometer and Agilent 2100 Nano were used to determine the RNA concentration and integrity.A 3 µg portion of total RNA for each sample was used for library construction by Majorbio Bio-Pharm Technology (Shanghai, China) using the Illumina TruSeq Small RNA Kit on an Illumina Novaseq XPlus platform.
Transcripts per million (TPM) reads were used to evaluate the relative expression levels of each miRNA, and DESeq2 software (v2.0.1.3)was used to identify the DEMs [83].The Bonferroni method was used to adjust the observed significance level.The miRNAs with at least a 2-fold change in expression and a p-adjusted of less than 0.05 were considered differentially expressed.

Quantitative Real-Time RT-PCR (qRT-PCR)
The leaves of PG and SG plants treated by low temperature for 0 h, 4 h, 8 h, 12 h and 16 h were used for qRT-PCR experiments.The expression levels of MELO3C002370, MELO3C009217, MELO3C018972, MELO3C016713, MELO3C012858 and MELO3C000732 were investigated in melon cultivars (TZ4, QM1, QM2, YZ9 and TM) at 8 h of normal (28 • C) and low-temperature (4 • C) treatment.For miRNA expression analyses, the total RNA was extracted by the miRcute miRNA extraction and separation kit (TIANGEN, Beijing, China), the first strand cDNA was synthesized by the miRcute Plus miRNA first strand cDNA kit (TIANGEN, Beijing, China) and the expression levels were detected by the miRcute Plus miRNA qPCR kit (TIANGEN, Beijing, China).For target gene expression analyses, the total RNA was extracted by the RNA Easy Fast Plant Tissue kit (TIANGEN, Beijing, China), the first strand cDNA was synthesized by the Hifiscript gDNA removable RT master mix (CWBIO, Taizhou, China) and the expression levels were detected by the 2 × SYBR Green qPCR Master Mix (UE EVERBRIGHT, Sayreville, NJ, USA).A CFX96 TouchTM qRT PCR assay system (BioRad, Hercules, CA, USA) was used for qRT-PCR experiments, and each miRNA or gene was analyzed with three biological and three technical replicates.SnRNA U6 and CmActin (MELO3C008032) of melon were used as the internal control.The relative expression levels of miRNAs and target genes were calculated by the 2 −∆∆ct method [85].Specific primers of miRNAs and target genes were designed by Primer Premier 5 and listed in Table S6.

Conclusions
In this study, phenotypic characteristics, physiological indexes, small-RNA sequencing and expression analyses were performed in SG and PG plants to explore the mechanism of changed low-temperature resistance by grafting in melon.Grafting with pumpkin rootstock could effectively improve the resistance of melon plants to low-temperature stress, certified by alleviated wilting, decreased MDA content, and increased SOD, CAT and POD activities.The results of small-RNA sequencing and qRT-PCR suggested that cme-miR156b, cme-miR156f and chr07-30026 acted as the positive regulators in resisting low-temperature stress and were significantly induced by cold in PG plants.Combined with target prediction and expression analyses, CmSPL13 (MELO3C002370), CmGCR2 (MELO3C016713) and CmRIC7 (MELO3C012858) were thought to play a negative role in the cold resistance of melon plants.These results provided a basic regulatory network between the key miRNAs and their target genes involved in cold tolerance and enriched the mechanism underlying how grafting improved the low-temperature resistance of melon plants.
Author Contributions: X.L., L.N. and W.Z. conceived and designed research.X.L. and X.Z.conducted most of the experiments.J.Z. performed data analysis.T.R. helped with qRT-PCR.X.L. and W.Z. wrote the manuscript.All authors have read and agreed to the published version of the manuscript.Data Availability Statement: All the experimental data are contained within this article or the Supplementary Materials.The small-RNA sequencing data for SG and PG plants are available in the Sequence Read Archive (SRA) database at the National Center for Biotechnology Information (https: //www.ncbi.nlm.nih.gov/(accessed on 4 November 1988)) under accession number PRJNA1090144.

Conflicts of Interest:
The authors declare no conflicts of interest.

Figure 1 .
Figure 1.Morphological and physiological characteristics of SG and PG plants under low-temperature stress.(a) Phenotypic changes in PG and SG plants at 4 • C. (b) Content of MDA.(c-e) Antioxidase activities of SOD (c), CAT (d) and POD (e).Lowercase letters represent the significant differences in physiological indexes at p < 0.05 (Student's t-test).

Figure 2 .
Figure 2. Small-RNA signatures of SG and PG seedlings under low-temperature stress.(a) Length distribution of sRNAs in twelve libraries.(b) Correlation between different samples.(c) Principal component analysis (PCA) of miRNA expression profiles.

Figure 4 .
Figure 4. Expression patterns of four key miRNAs under low-temperature stress.The blue (a-d) and yellow (e-h) columns represent the expression levels of miRNAs in SG and PG plants, respectively.Lowercase letters above the columns represent significant differences at p < 0.05 (Duncan's test).

Figure 5 .
Figure 5.Comparison of expression patterns between key miRNAs and predicted target genes.(a)LTR (yellow triangles) cis-acting element analysis of target genes.(b-q) Expression analyses of three miRNAs (b-d) and their target genes (e-q) in SG (blue columns) and PG (yellow columns) plants after 12 h of normal or cold treatment.Black triangles, circles and rectangles represent the genes targeted by cme-miR156b, cme-miR156f and chr07_30026, respectively.Lowercase letters above the columns represent the significant differences at p < 0.05 (Duncan's test).

Figure 6 .
Figure 6.Expression patterns of predicted target genes under low-temperature stress.Expression patterns of target genes in SG (a-f, blue columns) and PG (g-l, yellow columns) plants after lowtemperature treatment for 0 h, 4 h, 8 h, 12 h and 16 h.Lowercase letters above the columns represent the significant differences in transcript levels at p < 0.05 (Duncan's test).

Figure 7 .
Figure 7. Expression analyses of key target genes in five cultivars of melon with different lowtemperature resistance.(a,b) Phenotypic characteristics of five cultivars under low-temperature (4 • C) stress of 0 h (CK) (a) and 8 h (b).(c-h) Expression changes in six candidate target genes by lowtemperature stress in cultivars of melon.CT and LT represent the normal (28 • C) and low-temperature (4 • C) treatments, respectively.Asterisks represent significant differences at the transcriptional level (Student's t-test: *, p < 0.05; **, p < 0.01).

Funding:
This study was supported by the Hebei Agriculture Research System (Grant No. HBCT2023100208), and the Science and Technology Project of Hebei Education Department (Grant No. BJK2023111).

Table 1 .
Numbers of known and novel miRNAs in 12 libraries.